KR20020019938A - Vectors, Cells and Processes for Pyrimidine Deoxyribonucleosides Production - Google Patents

Abstract

Novel DNA constructs and host cells comprising the same are disclosed. DNA constructs comprise a transcription unit (e.g. operon) comprising DNA sequences encoding for ribonucleotide reductase and thioredoxin or a uridine kinase gene and/or a dCTP deaminase gene. In preferred embodiments the constructs comprising DNA sequences encoding for ribonucleotide reductase and thioredoxin further comprise DNA sequences encoding for thymidylate synthase and/or transcription units comprising sequences encoding for uridine kinase preferably together with dCTP deaminase. In particularly preferred embodiments, the host cells comprise constructs having all of the above characteristics wherein the host cell displays repressed or no uracil DNA glycosylase activity. This may be achieved by removal of the host cell ung gene. Use of host cells in the manufacture of pyrimidine deoxyribonucleotides e.g. thymidine is also disclosed.

Description

TECHNICAL FIELD The present invention relates to a vector, a cell, and a method for producing a pyrimidine deoxyribonucleoside for producing a pyrimidine deoxyribonucleoside.

Thymidine is useful as a pharmaceutical intermediate for the chemical synthesis of pharmaceutical intermediates, especially azidothymidine ("AZT", marketed under the trademark ZIDOVUDINE). ZIDOVUDINE-type AZT is the first therapeutic agent developed for HIV / AIDS, but its use is still important and widespread (Langreth, R., The Wall Street Journal, Nov. 21, 1995, pp B12]). ZIDOVUDINE-type AZT is particularly beneficial when used as a combination therapy, such as a combination with lamivudine (also known as 3TC) marketed under the trade name EPIVIR. A combination of AZT and 3TC is marketed under the trade name COMBIVIR. Since the HIV virus can mutate and thus be resistant to AZT or 3TC, the nucleotide-analog combination of the COMBIVIR-type is particularly useful because the reverse transcriptase can not necessarily have resistance to both of the two nucleoside analogs at the same time (Larder, BA et al., Science 269: 696-699, 1995). ZIDOVUDINE-type AZT is also useful when used in combination with drugs of the HIV protease inhibitor type (Waldholz, M., The Wall Street Journal, Jan. 30, 1996, pp B1) And it is possible to reduce the incidence of HIV infection at birth. In 1997, some 600,000 children died of maternal-infected AIDS at birth. Taking ZIDOVUDINE-type AZT for several months before giving birth can reduce the virus to 2/3. Thymidine for AZT production, produced by chemical synthesis, is very expensive.

U.S. Patent No. 5,213,972 (McCandliss & Anderson, hereinafter referred to as the "'972 patent") is incorporated herein by reference in its entirety for the production of pyrimidine deoxyribonucleoside (PdN) (Specifically, see Examples 7 to 14 of the '972 patent). A DNA sequence encoding a pyrimidine deoxyribonucleotide phosphohydrolase that converts a PdN monophosphate to a pyrimidine deoxyribonucleoside, and a replicable microorganism expressing it, is taught. More specifically, McCandliss & Anderson, supra, discloses a method for producing thymidine which can be used for thymidine production, including expressing deoxytimidylate phosphohydrolase (dTMPase) from Bacillus bacteriophage PBS1 A fermentation method is described. In nature, it has been found that this type of enzyme is expressed by bacteriophages into which DNA is introduced into the DNA, such as deoxyuridine or hydroxymethyldeoxyuridine, instead of thymidine.

In the thymidine fermentation process described in the '972 patent, enzymes that degrade thymidine (thymidine phosphorylase and free dephosphorylase) are mutagenized to accumulate thymidine. Therefore, the use of the dTMPase enzyme can assist in the generation of pathways enabling thymidine synthesis. However, the sole expression of dTMPase can not produce thymidine at a commercially viable level. Therefore, efforts to improve the production of thymidine to a commercially feasible level by the fermentation method using cells expressing dTMPase are still required to reduce the production cost as compared with the current chemical synthesis method.

For example, the biochemical pathway for producing pyrimidine deoxynucleotides in E. coli is highly regulated not only at transcriptional and translational levels, but also at the protein level by mechanisms such as attenuation, feedback inhibition and enzyme activation Escherichia coli and Salmonella Cellular and Molecular Biology ", Second Edition, Vol. 1, pp 580-599 [NeuHard, J. and RA Kelln, Biosynthesis and Conversion of Pyrimidines, Chapter 35 [In] Neidhardt, FC et al. , ASM Press, Washington DC, 1996). dTMPase and inhibits thymidine degradation by deo A (thymidine phosphorylase), udp (free phosphorylphosphorylase) and tdk (thymidine kinase) gene mutations and its expression products, But it is not commercially viable.

SUMMARY OF THE INVENTION

The process of biosynthesis of purines and pyrimidines involves a common step of reducing ribonucleoside diphosphate (in some species, triphosphate) to the corresponding deoxy analog. In the overall process, one pair of hydrogen atoms provided by NADPH and H ⁺ is required to reduce the ribose moiety to 2-deoxyribose. However, the direct electron donor is not the NADPH but the reduced form of the thermostable protein, referred to as thioredoxin or glutaredoxin, and one or more other unidentified sources, which include the ribonucleotide reductase system of E. coli trx A (thioredoxin) grx ( glutaredoxin ) double mutants [Neuhard and Kelln, supra]. The reducing equivalent of the reduced thioredoxin is delivered to the ribonucleoside diphosphate reductase that performs the reduction process. For example, manipulation of the above steps may be useful in improving the commercial production of purines and pyrimidine deoxynucleosides.

It is an object of the present invention to provide novel DNA constructs, such as vectors for use in producing pyrimidines and purine deoxynucleosides in a particularly recoverable amount, specifically in a commercially useful amount, and genetically modified microorganisms .

It is also an object of the present invention to provide an improved method than that described in McCandliss and Anderson, supra.

One aspect of the present invention provides a DNA construct comprising a ribonucleotide reductase gene and a transcription unit comprising a thioredoxin gene or a free dein kinase gene and / or a dCTP deaminase gene.

In one embodiment, the DNA construct comprises a transcription unit comprising a ribonucleotide reductase gene and a thioredoxin gene.

In another embodiment, the DNA construct comprises a transcription unit comprising a freezing kinase gene and / or a dCTP deaminase gene.

Preferably, the DNA construct comprises a transcription unit comprising a freezing kinase gene and a dCTP deaminase gene.

Most preferably, the DNA construct comprises a ribonucleotide reductase gene and a transcription unit comprising a thioredoxin gene and a free dein kinase gene and a dCTP deaminase gene.

Another aspect of the invention provides a modified host cell comprising a DNA construct according to the invention.

Another aspect of the present invention provides a method for producing pyrimidine, such as purine or thymidine, comprising using a culture medium comprising the modified host cell of the present invention and the modified host cell.

In one embodiment, the host cell is preferably a DNA transcription unit comprising a DNA sequence encoding ribonucleotide reductase and thioredoxin, which are less susceptible to allosteric repression than wild-type host cell equivalents or counterparts (A) (preferably heterologous to a host cell equivalent), a DNA sequence encoding a thymidylate synthase, and preferably a DNA sequence comprising the DNA construct (e. G., An operon) (E. G., An operon) located in the DNA construct, (b) a DNA sequence encoding a freezing kinase and preferably a dCTP deaminase, preferably a transcription unit located on the DNA construct , Operon), and (c) inhibited or deficient uracil DNA glycosylase activity.

In another embodiment, the DNA construct for use in producing a pyrimidine and derivatives thereof, particularly a pyrimidine deoxyribonucleoside, such as thymidine, in a recoverable amount comprises a free dein kinase and / or a dCTP deaminase (E. G., Operon) comprising a DNA sequence (preferably heterologous) that encodes a protein (e.

Also provided is a culture medium comprising genetically modified host cells comprising said constructs and expressing said host cells and said modified host cells.

Such aspects include, in part, DNA encoding the free tyrosine kinase and / or dCTP deaminase and, optionally, a host that further comprises a gene required for thymidine production, as proposed in U.S. Patent No. 5,213,972 Lt; RTI ID = 0.0 > thymidine < / RTI > production in the cell is significantly improved.

Aspects of the present invention are directed to providing improved DNA constructs for use in the commercial production of pyrimidine deoxyribonucleosides, particularly thymidine, and host cells comprising such constructs, as described in U.S. Patent No. 5,213,972 The first step is to make a number of improvements.

Other objects, features and advantages of the present invention will become apparent from the following description. It should be understood, however, that this is a preferred embodiment of the present invention and is for illustrative purposes only. It will be apparent to those skilled in the art that various modifications and variations are possible within the spirit and scope of the invention.

Preferred embodiments of the present invention

The constructs of the invention may be on a chromosome, or more preferably, extrachromosomal, e.g., on a vector.

The vector of the present invention may be a plasmid, a virus, a transposon, a mini chromosome or a phage, and preferably a plasmid. The vector containing the transcription unit may be introduced into the host cell according to any conventional method known to those skilled in the art, such as P1 transgenesis, electroporation, or transformation. Suitable host cells useful in the present invention include eukaryotic cells and prokaryotic cells (e. G., Bacteria). In prokaryotes are E. coli, Salmonella (Salmonella), Pseudomonas (Pseudomonas), Bacillus (Bacillus) and its mutant strains or the like. Due to the diversity of available mutant alleles, genetic tools, and information, E. coli is preferred. It is particularly preferred that the transduction method is available in the selected host cell so that the mutation can be easily transferred from one host cell to another host cell and that genetic mutation of the host is facilitated without direct mutagenization whenever new mutations are desired.

The present inventors have found that the use of bacteriophage T4 nrd A, nrd B and nrd C genes is particularly useful for reductase and thioredoxin coding in E. coli (Sjoeberg, BM et al., EMBO J., 5: 2031 Chem. 263: 16242-16251 (1988), and LeMaster, DM, J. Virol. 59: 759-760 (1986)). More specifically, since the T4 ribonucleotide reductase can not utilize E. coli thioredoxin, the T4 bacteriophage nrd A and nrd B genes encoding the ribonucleotide reductase are cloned together with T4 nrd C encoding T4 thioredoxin Lt; RTI ID = 0.0 > E. coli. ≪ / RTI > T4 ribonucleotide reductase has been found to be relatively insensitive to control by allosteric inhibition in vitro when compared to E. coli enzymes (Berglund, O., J. Biol. Chem. 247: 7276-7281, 1972) ). For example, unlike E. coli enzymes (Berglund, O., J. Biol. Chem. 247: 270-7275, 1972), the T4 ribonucleotide reductase is not inhibited by dATP but is effectively stimulated by dATP and ATP (Berglund, O., J. Biol. Chem. 247: 7276-7281, 1972).

The DNA sequence encoding the ribonucleotide reductase (for example, the nrd A and nrd B genes) and thioredoxin (for example, the nrd C gene) is preferably heterologous to the host cell DNA, preferably T (Preferably E. coli T bacteriophage), in particular T " even " phage, such as T2, T4 or T6 (Campbell, AM, Bacteriophages, Chapter 123, In Neidherdt, See, for example, Mathews, CK et al. (Eds.) Bacteriaophage T4, American Society of Microbiology, Washington DC, 1983). The term "derived from" is intended to define not only the source of its physical origin but also the material whose structural and / or functional properties correspond to the material from which it originated.

Another useful property of the T even-phage enzyme is its substrate specificity. Since K _m for UDP of a common E. coli ribonucleotide reductase is about 10 times higher than that for CDP (Neuhard and Kelln, supra), this enzyme uses only a small amount of UDP as a substrate. However, T4 enzyme is only the only difference between twice the K _m of the substrate CDP and UDP from the two paths for synthesizing dUTP (literature [Berglund, O., J. Biol Chem 247:.. 7276-7281, 1972 ]). Although attempts have been made to obtain functional expression of the T4 ribonucleotide reductase in E. coli, these previous efforts have proven successful only in separate expression of the components and only by in vitro mixing (Tseng, M. et al. -J., P. He, JM Hilfinger, and GR Greenberg, J. Bacteriol. 172: 6323-6332, 1990). Without wishing to be bound by theory, we believe that the expression of the T4 ribonucleotide reductase in E. coli is fatal because it is presumably free of the usual feedback inhibition pattern, so that it should be meticulously conditioned. Also contemplated are genes encoding a reductase and / or thioredoxin in the form of a precursor that will be processed to produce a mature form. The processing can be carried out through various intermediate forms.

The vector of the present invention preferably comprises a regulatory element (e. G., A promoter such as a lambda P _L , an operator, an activator, a repressor such as a lambda repressor, (Or enhancer), appropriate termination sequences, initiation sequences and ribosome binding sites. The vector may further comprise a selectable marker. Alternatively, regulatory elements (particularly lambda repressors) may be located on the host cell chromosome. it is desirable that nrd A and nrd B are arranged in the vector downstream of nrd C (in terms of the leading frame). In particular, it is preferable that nrd B is arranged downstream of nrd A. Thus, the most preferred arrangement is a vector comprising an operon comprising an nrd CAB.

The T4 ribonucleotide reductase lacks in vivo feedback control (J. Ji, RG Sargent, and CK Mathews, J. Biol. Chem. 266: 16289-16292, 1991), and Berglund, literature]). For example, in order to promote reduction of ribonucleoside diphosphate for thymidine production, nrd A, which is a gene encoding a regulatory subunit, is modified by a mutagenic approach or the like, for example, Or inhibition by allosteric inhibition, such as by inhibition by products from downstream events, can be produced.

In order to produce a T4 nrd A mutant, a site-directed mutagenesis may be used to modify or alter the gene base to encode, for example, the amino acid expected to alter the dTTP binding site involved in allosteric modulation For example, substitution). Amino acid sequence analysis of the T4 ribonucleotide reductase revealed a fragment that appears to be in good agreement with the putative consensus sequence believed to be involved in dTTP binding (EM Mcintosh and RH Haynes, Mol. Cell. Biol. 6: 1711-1721 , 1986). Several changes can be made using oligonucleotide directed mutagenesis in this region within the T4 ribonucleotide reductase. A common approach is the attempt to reduce the dTTP binding of the deoxycytidylate deaminase (More et al., JT, JM Ciesla, L.-M. Changchien, GF Maley and F. Maley, Biochemistry 33: 2104-2112, 1994). Mutations from T4 nrd A to ⁷⁹ Ila of Ile are considered to be very useful. For example, when strains containing mutations from T4 nrd A to Ile of ⁷⁹ Ala were fermented in shake flasks, thymidine productivity was significantly increased. As demonstrated below, the present inventors increased thymidine productivity by 25% over parent strain without this single change.

⁷⁹ mutation of Ala to Ile is a successful example, but one skilled in the art will know that the desired effect can be achieved by destroying only the dTTP binding and altering many other amino acids in this region so that the basic functionality of the enzyme is not destroyed. For example, ⁷⁹ Ala can be replaced with another amino acid having a side chain similar to Ile (e.g., leucine, valine). It is also contemplated that the position 79 in the region predicted to be a consensus region may be modified by another modification method (for example, a mutation method). It is another approach available to those skilled in the art to delete one or more amino acid positions within the consensus region and introduce synthetic DNA into that region.

In another aspect of the present invention, a construct comprising a transcription unit comprising a DNA sequence encoding a heterologous ribonucleotide reductase and a thioredoxin which are less susceptible to allosteric repression than wild-type host cell equivalents or counterparts For example, a vector). It will be clear to those skilled in the art that it is conventional experimentation and observation to determine the relative susceptibility to allosteric inhibition of a candidate heterologous reductase relative to wild-type host cell equivalents.

Transcription units containing DNA sequences such as nrd A, nrd B and nrd C genes are preferably operons in which the nrd genes are arranged in a line. This allows the gene to be transcribed as a single mRNA transcript. In order to minimize unproductive energy consumption by the host cell and further minimize the size of the plasmid, the operon contains only the genetic sequence (including any regulatory elements or control elements) necessary for the coding of the reductase and thioredoxin desirable. To do this, it may be necessary to remove redundant DNA (e.g., a unique intron in the phage T4 nrd B gene, Sjoberg, BM., Et al., EMBO J. 5: 2031-2036, 1986).

In another preferred embodiment, the vector of the present invention for use in the production of thymidine and the like may further comprise a DNA sequence encoding a thymidylate synthase (e.g., td gene) (see, for example, Refer to Chu, FK et al., J. Bacteriol. 169: 4368-4375 (1987)), as described in Chu, FK et al., Proc. Natl. Acad. Sci. USA 81: 3049-3053 ). The purpose of using the enzyme is to improve the control of the level of deoxyuridine produced, especially deoxyuridine impurity levels to thymidine. The dTMPase enzyme is not completely specific for dTMP. If K _m for dUMP is higher than for dTMP, PBS 1 dTMPase will produce deoxyuridine using dUMP as a substrate (Price, AR, Methods in Enzymol. 51: 285-290, 1978) . Deoxyuridine causes significant problems with thymidine tablets. Therefore, one way to reduce deoxyuridine production is to increase the level or efficiency of the thymidylate synthase, effectively converting dUMP to dTMP so that the internal concentration of dUMP is always kept at a very low level.

The thymidylate synthase gene ( td ) may be heterologous to the host cell, and the td is preferably derived from a T bacteriophage, such as T " even " phage (as defined above) td . td can be located in its native transcription unit, but it is preferred that td is located in the same transcription unit as the nrd gene, e. g., the same operon. It is also preferred that td is located downstream of the same operon (from the side of the reading frame) from the nrd gene.

[McCandliss and Anderson, supra] amplified the E. coli thymidylate synthase gene in plasmids pCG138 and pCG148 (see Table 5 of the '972 patent), which proved to be partially effective in reducing deoxyuridine. T4 thymidylate synthase is even more effective, since the E. coli enzyme is not controlled by any type of allosteric regulation [Neuhard and Kelln, supra], which is surprising in light of the fact. K _m 4 μM of the E. coli enzyme for dUMP (literature [Wahba, AJ and M. Friedkin, J. Biol Chem 237:. 3794-3801]) and the enzyme T4 for dUMP 2.73 μM K _m (literature [Maley, F , L. LaPat-Polasko, V. Frasca and GF Maley, Functional domains in T4 Thymidylate Synthase as probed by site-directed mutagenesis, Chapter 29 [In] Karam, JD [ed.] "Molecular Biology of Bacteriophage T4", American society for Microbiology, Washington DC, 1994, pp 322-325) are similar, and thus can not account for such a large difference in efficiency. Although not wishing to be bound by theory, the present inventors have found that E. coli thyA is an upstream gene (Bell-Penderson, D, JL Galloway Salvo, and M. Belfort, J. Bacteriol. 173: 1193-1200, 1991). ≪ / RTI >

In another preferred embodiment, host cells of the invention for use in the commercial production of pyrimidine deoxyribonucleosides, particularly thymidine and the like, include, for example, the udk gene encoding a free dein kinase, (e.g., operon) comprising a DNA sequence such as a dcd gene that encodes a dCTP deaminase (see, for example, Wang, L. and B. Weiss, J. Bacteriol. 174: 5647-5653 (1992), and Neuhard, J. and L. Taro, J. Bacteriol. 175: 5742-5743)

The constructs of this aspect of the invention are preferably heterologous to the host cell DNA, preferably a E. coli bacteriophage, especially a ribonucleotide reductase ( nrd A) derived from T " even " phage, such as T2, T4 or T6 And nrd B) and thioredoxin ( nrd C), or a precursor form thereof.

The uridine kinase produces UMP and CMP from uridine and cytidine using GTP (or dGTP) as a phosphate donor. This reaction is inhibited by UTP and CTP (J. Neuhard and RD Kelln, Biosynthesis and Conversions of Pyrimidines, Chapter 35 [in] FC Neidhart et al. [Eds], " Escherichia Coli and Salmonella Cellular and Molecular Biology" , Second Edition, ASM press, Washington DC). The present inventors have found that the use of a free dein kinase, particularly in conjunction with a dCTP deaminase, results in a marked improvement in the production of thymidine by the host cells that introduced such a change, with the teachings of '972 summarized above. Based on current information, this observation was entirely unexpected, as the free dein kinase does not have a direct role in the de novo biosynthesis of pyrimidines, and its use is limited to pyrimidine deoxyribonucleosides It would be beneficial for the commercial process for seed production. The udk and dcd genes are preferably arranged in a line in the same operon. Also contemplated are udk and dcd genes that encode the precursor forms that will be processed to produce the mature form. The processing can be carried out through various intermediate forms. The udk and dcd genes may be introduced into constructs (e.g., vectors) from any suitable source, according to methods known to those skilled in the art, such as P1 transgenesis, electroporation, or transformation.

Yurassil DNA glycosylase, an enzyme encoded by the ung gene, is responsible for the degradation of DNA containing yurassyl instead of thymidine. When the host cell of the present invention is used for commercial production of thymidine and the like, the intracellular concentration of dTTP can be reduced by using dTMP (a precursor of dTTP) for thymidine production. Therefore, the inventors have realized that the tendency to introduce uracil into potentially more potent host DNA can be fatal to wild-type hosts due to uracyl DNA glycosylase activity, which results in too much single-stranded breakage of the host cell DNA . Therefore, host cells useful in the present invention may also be those in which Elacsil DNA glycosylase activity is inhibited (as compared to unmodified cells) or not at all. Inhibition or absence of such activity may be achieved through a variety of methods which will be apparent to those skilled in the art. One example of such an approach is to introduce antagonists of the functional enzyme (or its precursor) into host cells to utilize (overall or partial) antagonism of the ung gene expression product. Other approaches include, for example, to modify the regulatory elements of the ung gene expression or ung gene that by itself, introducing a mutation in the expressed product of the ung gene uracil DNA glycoside sila the protein and (or) activity is little or no so and manipulating ung gene expression. Another approach is to delete the ung gene (or a portion thereof functionally important) from the host cell DNA. Without further manipulation, the host cell may be characterized by having no or low levels of uracil DNA glycosylase activity.

In a preferred embodiment of the present invention, the teachings herein are each advanced and introduced into host cells. The nrd , td , udk, and dcd genes may be located on separate structures, but all of them are preferably located on the same structure, e.g., on the same vector. Therefore, in a particularly preferred embodiment of the present invention, a modified ribozyme (preferably a T even-phage, e.g., of T4) that is less susceptible to allosteric inhibition than a wild-type host cell equivalent or counterpart A DNA construct comprising a DNA transcription unit (e. G., An operon) comprising a DNA sequence encoding a nucleotide reductase and thioredoxin and comprising a DNA construct further comprising: Or suppressed < / RTI > host cells. (a) a transcription unit (e. g., an operon) that encodes a thymidylate synthase (preferably heterologous to a host cell equivalent or counterpart), and (b) a free tyrosine kinase and preferably a dCTP deaminase A transcription unit (e. G., An operon)

The host cells modified according to the invention are particularly useful for the commercial production of pyrimidine deoxynucleosides. Particularly advantageous of the present invention is the ability to commercially produce thymidine using E. coli host cells containing (containing) a plasmid modified according to the present invention (particularly with the teachings of the '972 patent). Thus, modified host cells according to the present invention may further comprise dTMPase derived from PBS1 and the like and mutants taught in the '972 patent, such as deo A, tdk- 1, and udp- 1.

Typically, a fermentation process is used that involves culturing the cells in a culture medium in a suitable container. After culturing under appropriate conditions, the produced thymidine is collected and purified (enriched), optionally purified to pharmaceutical grades according to standard protocols. The purified thymidine can then be used to produce medicines, such as pharmaceutical compositions, such as AZT.

The present invention relates to a method for producing derivatives thereof, such as pyrimidines, purines, and deoxyribonucleosides, using genetically modified cells comprising novel DNA constructs.

The invention will now be described, by way of example only, with reference to the following figures:

Figure 1 schematically shows the production path of pCG366. It should be noted that the plasmid is not drawn at a constant rate.

Fig. 2 schematically shows production routes of pCG374 and pCG375.

Figure 3 shows a map of the plasmid pCG532.

4 is a recombinant E. coli strain CMG2451 (Ung ⁺⁾ and CMG2492 (Ung ^-) containing the plasmid pCG366 (nrd CAB td) according to Example 10 it shows the culture and thus thymidine production by the.

Figure 5 shows thymidine production by E. coli CMG2451 (pCG532) in a 30 L fermentor.

6 shows TdR and UdR (mg / L) obtained according to the purification protocol of Example 12. Fig.

Example 1

T4 nrd Cloning and activation of CAB gene

The bacteriophage T4 nrd AB gene was cloned by performing polymerase chain reaction (PCR) with isolated phage T4 DNA. The PCR primers of the nrd A gene

5'-TAT TCT AGA CGA TTT TCA AGT TGA GGA CTT ATG C-3 '(SEQ ID NO: 1); And

5'-TAT ATC GAT AAT TCA TTA CAA TTT ACA CGC TGC AC-3 '(SEQ ID NO: 2)

Respectively.

Restriction site XbaI was introduced at the start of amplified DNA and ClaI was introduced at the 3 'end of nrd A. The PCR amplification primers of the nrd B gene

5'-TAT ATC GAT AAA TGT AAA TTT AAG GAT TCT AAA TG-3 '(SEQ ID NO: 3) and

5'-TAT GTC GAC TCC TTA AAA GTA TTT TTT AAA ACT C-3 '(SEQ ID NO: 4)

Respectively.

Thus, the restriction site ClaI was introduced at the start site of nrd B and ApaI was introduced at the nrd B end of the amplified DNA. PCR fragments were cloned as shown in Figure 1 according to techniques known in the art. The cloned nrd AB gene was confirmed by enzyme activity assay. Cloned T4 gene in pKC30 nrd C to prepare a plasmid and pDL51 [LeMaster, DM, J. Virology 59 : 759-760,1986], T4 nrd C gene is di. Were obtained from D. LeMaster (Dept of Biochem., Univ., Wisconsin, Madison, Wis.). The gene was subcloned into a plasmid containing the nrd AB gene shown in Fig. The source and basic information of the starting materials used in Figure 1 are shown in Table 1.

PCG198 and pCG301 were prepared using a synthetic transcription terminator (see Figure 1 and Table 1). Specifically, the plasmid pBC sk ⁺ purchased from Stratagene (La Jolla, CA) was digested with restriction enzymes ApaI and Asp718I. Subsequently, the ECOTGP transcription termination sequence [d'Aubenton Carafa et al., J. Mol. Biol. 216: 839-843,1990] was ligation to replace the original sequence between the ApaI and Asp718I endonuclease sites. The fragment regenerated the ApaI recognition sequence in a new plasmid but removed Asp718I. The inserted DAN has the following composition generated from two oligonucleotides.

5'-CGAGC CCGCCTAATG AGCGGGCTTT TTT TT-3 '(SEQ ID NO: 5)

3'-CCGGGCTCG GGCGGATTAC TCGCCCGAAA AAAAACATG-5 '(SEQ ID NO: 6).

As shown in Figure 1, the plasmid pCG198 was ligated with pCG173 containing the lambda P _L promoter cloned from plasmid pKC30 into pBluescript 11 ks (Stratagene, La Jolla, CA). Both pCB198 and pCG173 were digested with HindIII and AsnI to generate a new plasmid pCG301 containing the lambda P _L promoter, a number of restriction enzyme cloning sites followed by an ECOTGP terminator sequence copied from the tryptophan operon leader peptide region.

The T4 ribonucleotide reductase activity was determined by HPLC in a manner that does not use radioactive isotopes and UDP substrates. Direct ribonucleotide reductase assay methods included 1 mM NADPH, 1 mM DTT, 0.5 mM dATP, 0.6 mM UDP, 20 mM Tris (pH 8.0) and 5 mM MgCl ₂ . Under these conditions, the E. coli ribonucleotide reductase was inhibited and was not detected. Enzyme reaction (100 ㎕) was stopped by adding 10 50 of 50% trichloroacetic acid (TCA). After 10 minutes on the ice sheet, the sample was centrifuged with a microcentrifuge. The supernatant was extracted 4 times with diethyl ether to remove TCA. After adding 5 ml of Tris buffer (1.0 M pH 8.0), 2 占 of a 40 mg / ml rattle snake venom (Sigma) was added and the sample was incubated at 37 占 폚 for 60 minutes. Subsequently, the sample was heated at 70 DEG C for 3 minutes and centrifuged for 5 minutes to remove the precipitate. The volume was homogenized and analyzed with HPLC equipped with a UV detector and with deoxyuridine as a standard. The column was a Spherisorb ODS-2, 5 micron, 250 mm x 4.6 mm using a 12 mM ammonium phosphate (pH 5.0) mobile phase and a 1.0 ml / min flow rate. The results are shown in Table 2, demonstrating that the cells containing the plasmid pCG343 functionally expressed the T4 ribonucleotide reductase.

Ribonucleotide reductase activity Strain Induction condition Inactive nmol / 10 min / mg protein CMG1093 Non-Diagram 0 CMG1093 Judo 0 CMG1093 / pCG343 Non-Diagram 0 CMG1093 / pCG343 Judo 704.7

Example 2

Induction of host strain CMG2451 from strain CMG1115

The strain CMG1115 is fully described in McCandliss & Anderson (US Patent No. 5,213,972). CMG1115 was used as a starting point for the invention described herein. Strain CMG1115 was selected for growth in a 30 mg / l 5-fluoroureidane containing medium to obtain thymidine productivity, resulting in strain CMG2401. The strain CMG2401 was then screened for growth in a 30 mg / l 3'-azido-3'-deoxythymidine containing medium to obtain strain CMG2404. CMG2404 requires L-proline at growth due to inherited mutant < RTI ID = 0.0 > (lac-pro) < / RTI > inherently inherited from parental strain JM101. CMG2404 and Hfr strain CAG5053 (Singer, M. et al. Microbiological Review: 5: 1-24, 1989) were Hfr crossed according to techniques known in the art to obtain Lac ⁺ , Pro ⁺ , strain CMG2434. The udp (free phosphorylphosphorylase) mutation of CMG2434 still partially has a free phosphorylphosphorylase activity, which is proven on the basis of thymidine accumulation after inducing thymidine production. The udp mutation can be reintroduced from the CGSC5128 (E. coli Genetic Stock Center, Yale University) by phage P1 transfection according to the known art in the art. First, from strain CAG18491 to met E3079: : Tn10 is transfected with CMG2434 and used as a positive selection marker for the transduction of udp . Subsequently, udp- 1 is transformed with the met E3079 :: Tn10 derivative of strain CMG2434 and grown without L-methionine in a given medium The udp- 1 derivative was named strain CMG 2451. The lineage of CMG 2451 is summarized in Table 3.

Genealogy of E. coli host strain CMG2451 Strain genotype Derived from ^a CMG1115 CMG1116 (Tn5 :: dTMPase kan ^R ) Insert Tn5 :: dTMPase from G132 CMG2401 CMG1115 FUdR ^R 5-fluoro-2 ' deoxyindoline resistance CMG2404 CMG2401 AZT ^R 3'-azido-3 'deoxythymidine resistance CMG2434 CMG2404 Lac ⁺ Pro ⁺ CAG5053^bAnd △ (lac-proAB) CMG2448 CMG2434 met E3079 :: Tn10 From CAG18491 ^b met E3079 :: Tn10 CMG2451 CMG2448 udp-1 metE ⁺ tet ^s From CGSC5128 ^c udp -1 and an alternate met E3079 :: Tn10

a. In some cases, mutations were introduced into E. coli strains by phage P1 transduction. If the mutation is a selectable marker, it was used directly for P1 transduction. If the mutation is not a selectable marker, Tn10 is inserted adjacent to use P1 co-transduction.

b. Singer, M., et al. Microbiological Review: 53: 1-24, 1989]

c. All CGSCs. (E. coli Genetic Stock Center, Yale University, P.O. Box 208104, New Haven, CT 06520-8104).

Example 3

Cloning and Expression of T4 td Gene into Thymidine Production Plasmid

T4 < / RTI > td gene with a 1017 base pair intron (West et al. (J. Biol. Chem 261: 13446-13450, 1986)]. The td gene was subcloned as a linker using two oligonucleotides of the following sequence:

5'-GAT CCG GAG GAT AAA TGA AAC AAT ACC AAG ATT TAA T-3 '(SEQ ID NO: 7); And

5'-TAA ATC TTG GTA TTG TTT CAT TTA TCC TCC G-3 '(SEQ ID NO: 8).

The resulting plasmid was pCG356. Subsequently, the td gene was subcloned into pCG343 from pCG356 to prepare pCG360 (Fig. 1). The tetracycline resistance gene and plasmid replication origin were subcloned into pCG360 from pBR322 (Bolivar, F. et al., Gene 2: 95-113, 1977) to form pCG366. The thymidylate synthase activity was measured by spectrophotometry of Wahba and Friedkin (J. Biol. Chem. 236: PC11-PC12,1961). The results are shown in Table 4.

Thymidylate synthase activity Strain Inactive < RTI ID = 0.0 > OD340 / mg < CMG1093 -0.72 CMG1093 / pKTdDI 3.24 CMG1093 / pCG356 3.45 CMG1093 / pCG360 1.43

Example 4

Trimidine production and T4 when deoxyuridine decline td Shaking flask data showing effect

The shim flask fermentation was used to evaluate the thymidine productivity of the different E. coli recombinants. The shaking flask fermentation broth and method used in this and other examples are described in Example 6 below. At this time, 20 ml volume per flask was inoculated with 2 ml of seed culture and cultured in a shaking incubator at 30 캜. When the OD 600nm reached about 10, the flask was transferred to a 37 ° C shaking incubator and the λ P _L promoter was induced mildly for 30 minutes. Then, the flask was transferred to a shaking incubator at 35 캜 to continue fermentation. Glucose was added during fermentation as needed and titrated to pH 7 with ammonia according to phenol red color. The thymidine concentration was determined by HPLC using a Spherisorb ODS-2, 5 micron, 250-mm x 4.6 mm column, and 25 mM ammonium phosphate (pH 3.3) mobile phase, flow rate of about 1.5 ml / min.

The strain CMG2451 / pCG366 (T4 nrd CAB, td ) was compared with CMG2451 / pCG343 (T4 nrd CAB) in shake flask fermentation. Table 5 shows that deoxyuridine has been reduced in concentration and that deoxyuridine has been converted to thymidine due to the T4 td gene in strain CMG2451 / pCG366.

Effect of thymidine synthesis on thymidine production in 66 hours Strain Thymidine (mg / l) Deoxyuridine (mg / l) % Deoxyuridine CMG2451 / pCG343 1198 1728 144.2 CMG2451 / pCG366 3327 206 6.2

Example 5

⁷⁹ Production of T4 ribonucleotide reductase mutants from Ala to Ile

The mutation is described in Kunkel, TA, Proc. Natl. Acad. Sci. USA, 82: 488-492, 1985) using a site-directed mutagenesis method. All materials used to make mutants such as pTZ18U phagemid DNA, M13KO7 helper phage, bacterial strains E. coli CJ236 and MV1190, T7 DNA polymerase and T4 DNA ligase were purchased from Bio-Rad Laboratories, Hercules). ≪ / RTI > First, an Xbal / HindIII DNA fragment containing the nrd A gene of T4 bacteriophage was isolated from the plasmid pCG312 (ChemGen Corp., Table 1). The XbaI / HindIII DNA fragment was ligated to the XbaI / HindIII site of the pTZ18U phagemid vector using standard protocols (Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning, Cold Spring Harbor Laboratory, New York, Respectively. The phagemid pCG464 containing the insert was introduced into E. coli CJ236. The strain lacks dUTPase ( dut ) and uracil-N-glycosylase ( ung ) and substitutes thymidine with uracil in newly synthesized DNA. The single stranded DNA of pCG464 containing eucalyptus was isolated from CJ236 according to the method of using bio-Rad Laboratories. The DNA (0.2 pMole) the original object ⁷⁹ mutation encoding an Ile in place of Ala (underlined) of phosphorylated primer containing the sequence 5'-AGC AAA CAT TAA ACA GCG TGC AAT TAC ATA TTG ATA ATC AGG TTC-3 ' (SEQ ID NO: 9) and annealed with 6 pMole.

Complementary strand DNA was synthesized using T7 DNA polymerase as described in the Bio-Rad protocol. The reaction product was transformed into Escherichia coli MV1190 containing wild-type eucalyptin-N-glycosylase that cleaves the parent strand containing uracil to increase the mutant strand. Direct DNA sequencing using a Silver Sequence DNA Sequencing System, purchased from Promega Corp, Madison, Wis., USA, identified plasmids containing the desired mutations. All four analyzed transformants were found to contain plasmids with mutations. One of them was named pCG492 and used in the subsequent experiments. To confirm whether the mutation affects thymidine synthesis, the production plasmid pCG366 (ChemGen Corp., Table 1 and Figure 1) was introduced. To this end, the KpnI / AflII DNA fragment of pCG366 containing the 5'-portion of the nrd A gene was replaced with a KpnI / AflII fragment from pCG492 containing the mutation. A new plasmid pCG494 was introduced into the production strain CMG2451 (ChemGen Corp., Example 2 and Table 4 above). The effect of the T4 nrd A mutation was assessed in comparison with thymidine production by CMG2451 (pCG494) and CMG2451 (pCG366) shown in Example 6 below.

Example 6

Shaking flask fermentation for thymidine production using CMG2451 (pCG366) and CMG2451 (pCG494)

A 250 ml baffle flask containing 25 ml of production medium was inoculated with 2 ml of freshly cultured seed culture in LB broth supplemented with an appropriate antibiotic. The culture was incubated at 30 rpm in a shaking incubator at 250 rpm. When the OD ₆₀₀ reached about 5, the flask was transferred to a 37 ° C shaking incubator for 30 minutes. The flask was then fermented continuously in a 35 DEG C shaking incubator. The composition (g / l) of the production medium is as follows: Ardamine YEP-S (Red Star Yeast & Products, Milwaukee, WI) -10; CaCO ₃ -10; MgSO ₄ -0.4; Phenol red -0.24; PP90BT (DMV International, Fraser, NY) -4.5; Sorbitol-20; Chloramphenicol-0.03; Trace element (1000x) -1 ml / l. The trace element (100Ox) composition was boric acid-0.05; Calcium chloride-20; Cobalt sulfate-0.05; Copper sulfate-0.01; Iron (I) -20 sulfate; Iron (II) -20; Magnesium sulfate-0.5; Sodium molybdate-0.1; And zinc sulfate-0.1. At the induction, 10 grams of adamin YEP-S per liter was added. Glucose was added on average every 2 hours (2.5 g / l) during fermentation. The pH in the flask was maintained at about 7.0 by addition of 4N NH ₄ OH to the phenol red indicator dye. The sample was diluted 1:10 in 10 mM H ₂ SO ₄ in a salt solution and OD ₆₀₀ was measured. The thymidine concentration was measured by reversed phase C-18 HPLC equipped with an Alltech Spherisorb ODS-2 column and a Shimadzu spectrophotometric detector 260 nm. The mobile phase was 25 mM NH ₄ H ₂ PO ₄ (pH 3.3) in water at a constant rate of 1.5 ml / min.

The results of thymidine production by CMG2451 (pCG366) and CMG2451 (pCG494) after 2, 17, and 25 hours after induction are shown in Table 6. This experiment was repeated with two flasks, and the difference between the two flasks did not exceed 15%. The results showed that the T4 nrd A mutant produced more of the wild-type nrd A strain. This was confirmed in several independent flask experiments using the same bacterial strain.

Thymidine production by CMG2451 (pCG366) and CMG2451 (pCG494) Strain 2 hours 17 hours 25 hours Inactivity (mg / l / OD) CMG2451 (pCG366) 6.1 32.5 43.1 CMG2452 (pCG494) 8.5 36.1 51.4

Example 7

E. coli for the production of thymine udk Shake flask fermentation to prove the effect of gene

The dcd gene or the dcd udk operon was cloned into the pACYC177 vector. The vector with the p15a origin of replication is different from the colEl based plasmid such as pCG366 and is therefore compatible and can be maintained in the same host containing the colEl based plasmid. Details of the construction of the plasmid producing pCG374 ( udk dcd ) or pCG375 ( dcd ) are shown in Fig. The genes on these plasmids are expressed from the E. coli promoter of the udk dcd operon.

Plasmids pCG374 and pCG375 were introduced into CMG2451 (pCG366) using the ampicillin resistance screening method and the effect on thymidine production was tested by shaking flask fermentation described in Example 6. The results at several points are shown in Table 7. Inactive (cell thymidine per OD) is however similar for cases that do not exist or if the udk gene is present, the was a cell having the udk gene on the second plasmid pCG374 growth at a density high cell density even more thymidine (dcd 5.8 g / l compared to 3.0 g / l for strains containing the second plasmid only). These results were unexpected, as it was unclear whether the uridine phosphorylase had this effect.

Based on the above data and other information, the udk gene was selected and introduced together with the E. coli dcd gene to prepare plasmid pCG532 (see Example 8 and Fig. 3).

Comparison of the effect of the second plasmid containing dcd or dcd udk on the basic thymidine production of CMG2451 (pCG366) Time (hr) O.D.600 Thymidine (mg / l) Bithimidine (mg / l / OD) Basic strains and plasmids CMG2451 (pCG366) CMG2451 (pCG366) CMG2451 (pCG366) CMG2451 (pCG366) CMG2451 (pCG366) CMG2451 (pCG366) A second pACYC 177 base plasmid pCG374 containing dcd udk pCG375 containing dcd pCG374 containing dcd udk pCG375 containing dcd pCG374 containing dcd udk pCG375 containing dcd 8 hr 29.112 29.94 889 884 30.5 29.5 18 hr 40.206 32.946 1721 1608 42.8 48.8 42 hr 48.348 33.96 4020 2740 83.1 80.7 66 hr 57.498 28.614 5804 3012 100.9 105.3

Example 8

E. coli to the production plasmid pCG494 udk dcd Cloning of operons

The udk and dcd genes of E. coli each encode a pyrimidine ribonucleotide kinase and a dCTP deaminase. Both genes were mapped to a 3.4 kb BamHI / PstI DNA fragment of the Kohara genomic library of Lambda phage 355 (Kohara, Y., Akiyama, K. and Isono, K., Cell 50, 495-508, 1987). udk dcd is shown to be present in the upstream and in the same direction of transcription dcd [Neuhard, J. and Tarpo, L., J. Bacteriol. 175: 5742-5743,1993]. The gene was cloned into the production plasmid in two steps.

First, a 3.4 kb BamHI / PstI DNA fragment of lambda 355 was cloned into a number of cloning sites of the plasmid pUC18 (Yamisch-Perron, C., Vieira, J. and Messing, J., Gene 33: 103-109,1985) pCG358). The fragment was then cleaved from the polylinker region of pCG358 with BamHI and SphI to replace the 0.7 kb BglII / SphI fragment (containing part of the tetracycline resistance gene) of the production plasmid pCG494. The final 14.7 kb plasmid, pCG532, contains the replication origin of the colE1-compatible family, the chloramphenicol resistant gene, the udk and dcd genes, and the T4 bacteriophage nrd CAB under the control of the P _L promoter of bacteriophage lambda (two of thioredoxin and ribonucleotide reductase Subunit respectively) and T4 td (a thymidylate synthase coding) gene. The T4 nrd A gene of pCG532 was first altered by site-directed mutagenesis (Ile at ⁷⁹ Ala).

The synthetic transcription terminator is located downstream of the td gene and prevents it from being transcribed and transcribed into the cloned region. The gene map of the plasmid pCG532 is shown in Fig.

Example 9

Thymidine shake flask fermentation by CMG2451 (pCG494) and CMG2451 (pCG532)

Plasmid pCG532 containing E. coli udk and dcd genes was introduced into the production strain CMG2451. The new strain (pCG494) was tested by sham flask experiments with parental strain CMG2451 (pCG494) to compare thymidine production. Two independent experiments are shown in Table 8. The first experiment was performed as described above and the sample was taken at 17 hours after induction. In the second experiment, cells were induced at high OD (about 9) and samples were taken at 3 hours after induction for analysis. In both cases, the strains containing the cloned udk and dcd genes produced more than the parent strains.

Fermentation of thymidine with CMG2451 (pCG494) and CMG2451 (pCG532) Experiment 1 Strain O.D 600 TdR (mg / l) Inactivity (mg / l / OD) CMG2451 (pCG494) 16.5 599 36.3 CMG2451 (pCG532) 17.5 867 49.5 Experiment 2 Strain CMG2451 (pCG494) 8.5 149 17.5 CMG2451 (pCG532) 8.4 192 22.8

Example 10

ung Addition of mutations and its effect on thymidine production on shake flask fermentation

Uracil DNA glycoside sila claim by using the above P1 transduction voice strain ung :: Tn1O (Varshney, U., et al, J. Biol Chem 263:... 7776-7784,1988) a mutation in the host CMG2451 And was named CMG2492. Plasmid pCG366 was introduced into CMG2492. A comparative experiment of Ung ^- and Ung ⁺ strains for thymidine synthesis in a shaking flask using the flask method described in Example 6 is shown in FIG. Cells were cultured in a 250 ml flask at 30 캜 and changed to 37 캜 to induce thymidine synthesis for 30 minutes and then changed to 35 캜. The Ung ^- host lacking the Elacsil DNA glycosylase has grown longer and thymidine is 30% more produced.

Example 11

Production of thymidine in a 30 liter fermentor using strain CMG2451 (pCG532)

The thymidine was produced in a 30 liter fermentor (B. Braun Biotec Biostat C) with strain CMG2451 / 532 using the following conditions. Seed cultures (500 ml) were cultivated in LB medium (5 g / l Difco yeast extract, 10 g / l Difco tryptone) supplemented with 30 mg / l chloramphenicol and 25 mg / l kanamycin , 5 g / l NaCl) at 30 [deg.] C until reaching OD 600 nm 2.37, final pH 6.69 in a 4-liter baffle shake flask.

The initial batches in a fermenter (12 liters) containing the compositions listed in Table 9 were sterilized at 121 占 폚 for 55 minutes. After cooling, autoclaved (500 ml) was added separately and the batch was adjusted to 20 g / l sorbitol and 3.0 g / l MgSO ₄ 7H ₂ 0. Sterile filtration solution (200 ml) was added to adjust the initial batch to 30 mg / l chloramphenicol, 25 mg / l kanamycin, 1 mg / l d-biotin, 10 mg / l thiamin and 10 mg / l nicotinic acid .

Three feed solutions were prepared: a) Cerelose 2001 (dextrose monohydrate) containing 2 mg / l biotin, 20 mg / l thiamine, 20 mg / l nicotinic acid and 30 mg / l chloramphenicol 562 g / l; b) 717 g / l of sorbitol containing 4 mg / l biotin, 40 mg / l thiamin, 40 mg / l nicotinic acid and 60 mg / l chloramphenicol; And c) a nitrogenous mixture containing 360 g / l Amberex 695 AG (Red Star Yeast & Products, Milwaukee, Wis., USA), 6 g / l PP90M (DMV International, Prasad, NY) and 0.1 ml / l Mazu DF1 OPMOD11 (BASF). The feed solution was sterilized for 40 to 50 minutes under 18 PSI vapor pressure.

The initial batch composition in a 30 ml fermenter ingredient Concentration (g / l or ml / l) Sodium hexametaphosphate 4 KH ₂ PO ₄ 4 (NH ₄ ) ₂ HPO ₄ 4 CaCl ₂ 0.4 Citric acid 0.5 Amberex 695 (Red Star Yeast & Products, Milwaukee, Wis., USA) 20 PP90M (DMV International, Prasse, NY) 15 Tryptone (Difco, Detroit, MI) 5 1000 x trace element (see Example 7) 1 ml / l CaCO ₃ 5 Glycine 1.83 Mazu DF10PMOD11 formulator (BASF 0.2 mL / l Specialty Products, Gurnee, IL) 0.2 ml / l

Operating conditions were initial temperature 31 ° C; RPM 600; Air flow 3 LPM; pH 6.8; The pressure was 0 Bar. Dissolved oxygen was adjusted to 25% saturation using an airflow rate control loop. The RPM was changed from 31 ° C to 35 ° C at 9.2 hours when the culture reached OD 37.8 at 750 RPM for 8 hours, 850 RPM for 9 hours, and increased to 950 RPM. Starting at 9.7 h, back pressure was applied to the culture using oxygen transfer at a maximum of 0.6 Bar. The rate of temperature change for the induction of thymidine synthesis was 0.2 [deg.] C per minute.

The cell mass was supplied to an 85 ml batch of sorbitol feed solution (b) at OD 600 nm (after dilution in 50 mM H ₂ SO ₄ to dissolve the salt) and after 20 cell masses increase OD 5, respectively . (A) of dextrose monohydrate (glucose) under the control of the DO PID control loop in a fermentor (B. Braun fermentor) set at 25% was stopped. Briefly, the feed was stopped when the dissolved oxygen was less than 25% and the sugar feed was started when the dissolved oxygen was greater than 25%. The protocol itself kept the glucose concentration low, but prevented the glucose from depleting the culture for very long periods of time. Triton (500 ml) was fed for 11 hours, 16.2 hours, 21.2 hours, 28.2 hours, 33.2 hours and 40.5 hours.

Cell mass, thymidine and deoxyindoline accumulation during fermentation are shown in Fig.

Example 12

Purification of thymidine from fermentation broth

Dowex Optipore L-285 (Dow Chemical Company) was suspended in deionized water and filled into a 48 mm diameter glass column to produce a bed volume of about 500 ml. The column was washed with 500 ml of 5% NaOH and washed with deionized water until the eluate had a pH of 7.0.

A 500 ml fermentation broth containing a thymidine (TdR) concentration of 4.890 g / l and a deoxyuridine (UdR) concentration of 1.040 g / l was loaded onto the column. The column was washed with two times the bed volume of deionized water. TdR and UdR were diluted with 5% alcohol solution (90.5% ethanol, 4.5% methanol, 5% isopropyl alcohol) twice the volume of the bed followed by 10% alcohol reagent twice the volume of the bed and 15% alcohol reagent twice the volume of the bed Lt; / RTI > The TdR and UdR in the 25 ml fractions are shown in Fig. The column was washed with 5% NaOH followed by deionized water to pH 7.0 to regenerate and the process was repeated. Fractions 91-160 were taken together from two separate runs and dried using a rotary vacuum evaporator. Thymidine was redissolved in a minimum amount of hot water and crystallized at 4 占 폚. The crystals were then recrystallized twice and dried in an oven at 55 ° C for 15 hours. A total of 979.8 mg of crystalline thymidine was obtained with a purity of more than 99% suitable for use as a pharmaceutical intermediate.

Adjacent fractions containing both deoxyuridine and thymidine were pooled together with the mother liquor and the alcohol was reduced with a rotary vacuum evaporator to a final volume of 1200 ml. The total amount of TdR in the 1200 ml solution was 3571 mg. The total recovery (including 3571 mg of the TdR adjacent fraction) was 93.1%.

Claims (48)

A DNA construct comprising a ribonucleotide reductase gene and a transcription unit comprising a thioredoxin gene or a free dein kinase gene and / or a dCTP deaminase gene. The DNA construct of claim 1, wherein the transcription unit comprises a ribonucleotide reductase gene and a thioredoxin gene. The DNA construct of claim 1, wherein the transcription unit comprises a free dinucleotide gene and / or a dCTP deaminase gene. The DNA construct of claim 1, wherein the transcription unit comprises a free dinucleotide gene and a dCTP deaminase gene. 5. A DNA construct according to any one of claims 1 to 4, which is a vector of extrachromosomal. 6. The DNA construct according to claim 5, wherein the vector is a plasmid, a virus, a transposon, a mini chromosome or a phage. 7. The vector according to claim 6, wherein the vector is a plasmid in which the ribonucleotide reductase gene is the nrd A gene and / or the nrd B gene, the thioredoxin gene is the nrd C gene, and the genes are arranged in one operon structure. The DNA construct according to claim 7, wherein the transcription unit comprises the nrd A gene and the nrd B gene. 9. The DNA construct according to claim 8, wherein the nrd A and nrd B genes are located downstream of the nrd C gene. The method of claim 8, nrd C gene and upstream of the nrd genes A, nrd A gene DNA construct in B upstream of the nrd genes. 7. The DNA construct of claim 6, further comprising a regulatory element. 12. The DNA construct of claim 11, wherein the regulatory element is selected from the group consisting of a promoter, an operator, a termination sequence, a start sequence and a ribosome binding site. 13. The DNA construct of claim 12, wherein the promoter is a lambda P _L promoter or derivative therefrom. 13. The DNA construct of claim 12, wherein the termination sequence is a synthetic terminator. 8. The method according to claim 7, wherein the ribonucleotide reductase encoded by the unit is selected from the group consisting of the nrd A gene and the nrd A gene so as to be less susceptible to allosteric repression than a ribonucleotide reductase encoded by a unit containing an unmodified nrd A gene Modified DNA structure. 16. The DNA construct according to claim 15, wherein the nrd A gene is modified at the dTTP binding site. 16. The DNA construct according to claim 15, wherein the nrd A gene is modified to code by changing Ala to Ile at position 79 of the nrd A expression product. 8. The DNA construct of claim 7, wherein the nrd A, nrd B and nrd C genes are derived from a T phage. 19. The DNA construct of claim 18, wherein the T-phage is a T even-numbered phage. 20. The DNA construct of claim 19, wherein the T even-phage is a T4 phage. The DNA construct according to any one of claims 1 to 4, further comprising a thymidylate synthase gene. 22. The DNA construct according to claim 21, wherein the thymidylate synthase gene is a td gene. 23. The DNA construct according to claim 22, wherein the td gene is located on the same vector as the nrd A, nrd B and nrd C genes. 23. The DNA construct according to claim 22, wherein the td gene is located on the same operon as the nrd A, nrd B and nrd C genes. 25. The DNA construct according to claim 24, wherein the td gene is located downstream (in terms of the reading frame) of the nrd A, nrd B and nrd C genes. 23. The DNA construct according to claim 22, wherein the td gene is derived from a T phage. 27. The DNA construct of claim 26, wherein the td gene is derived from a T even-numbered phage. 28. The DNA construct of claim 27, wherein the td gene is derived from a T4 phage. 3. The DNA construct of claim 2, further comprising a freezing kinase gene and / or a dCTP deaminase gene. 30. The DNA construct of claim 29, comprising both a free dein kinase gene and a dCTP deaminase gene. The DNA construct according to claim 1, 3, or 4, wherein the free dinin kinase gene is a udk gene. The DNA construct according to claim 1, 3 or 4, wherein the dCTP deaminase gene is a dcd gene. 32. A modified host cell comprising a DNA construct according to any one of claims 1 to 32. 34. The modified host cell of claim 33, wherein the yurasil DNA glycosylase activity is inhibited or not at all. 35. The modified host cell of claim 34, wherein at least one of the host cell DNA sequences encoding the Elacsil DNA glycosylase activity is modified to encode an expression product exhibiting inhibitory or low levels of Elacsil DNA glycosylase activity. 36. The modified host cell of claim 35, wherein the host cell DNA sequence is an ung gene. 35. The modified host cell of claim 34, wherein one or more of the host cell DNA sequences encoding eucalyptus DNA glycosylase activity is removed. 34. The modified host cell of claim 33, wherein the host cell is a eukaryotic cell or a prokaryotic cell. 39. The method of claim 38, E. coli, Salmonella (Salmonella), Pseudomonas (Pseudomonas), Bacillus (Bacillus) and a saccharide as MY access (Saccharomyces), a modified host cell, selected from the group consisting of. A DNA construct comprising a DNA transcription unit comprising a ribonucleotide reductase gene and a thioredoxin gene that are less susceptible to allosteric inhibition than a wild-type reductase equivalent, (a) a transcription unit that contains a thymidylate synthase gene that is heterologous to the thymidylate synthase gene of the host cell and that is located on the DNA structure, (b) a transcription unit that contains a freerin kinase gene and is located on the DNA construct, (c) a transcription unit comprising a dCTP deaminase gene located on said DNA construct, and (d) a modified host cell further comprising one or more characteristics of inhibited or deficient uracil DNA glycosylase activity. 41. The modified host cell of claim 40, wherein the ribonucleotide reductase gene is modified at the dTTP binding site. 42. The modified host cell of claim 41, wherein the ribonucleotide reductase gene is the nrd A gene, wherein said modification is changed from Ala to Ile at position 79 of the nrd A expression product. 39. The modified host cell of claim 38, wherein the DNA construct comprises both the liberin kinase gene and the dCTP deaminase gene. 41. The modified host cell of claim 40 comprising each of the features of (a) to (d). 44. A method for producing a pyrimidine deoxyribonucleoside comprising culturing a host cell according to any one of claims 33 to 44. 46. The method of claim 45, wherein the deoxyribonucleoside is thymidine. 44. A method for the production of agatimidins, comprising culturing a host cell according to any one of claims 33 to 44. 44. A culture medium comprising the host cell according to any one of claims 33 to 44.